Metabolites of N-(4-{[6,7-Bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N&#39;-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

ABSTRACT

The invention relates to metabolites of cabozantinib (I) as well as uses thereof.

PRIORITY CLAIM

This application claims priority to U.S. Ser. No. 61/792,413, filed Mar.15, 2013. The entire contents of the aforementioned application areincorporated herein.

TECHNICAL FIELD

This disclosure relates to metabolites ofN-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide,a c-Met inhibitor.

BACKGROUND

Traditionally, dramatic improvements in the treatment of cancer areassociated with identification of therapeutic agents acting throughnovel mechanisms. One mechanism that can be exploited in cancertreatment is the modulation of protein kinase activity because signaltransduction through protein kinase activation is responsible for manyof the characteristics of tumor cells. Protein kinase signaltransduction is of particular relevance in, for example, thyroid,gastric, head and neck, lung, breast, prostate, and colorectal cancers,as well as in the growth and proliferation of brain tumor cells.

Protein kinases can be categorized as receptor type or non-receptortype. Receptor-type tyrosine kinases are comprised of a large number oftransmembrane receptors with diverse biological activity. For a detaileddiscussion of the receptor-type tyrosine kinases, see Plowman et al.,DN&P 7(6): 334-339, 1994. Since protein kinases and their ligands playcritical roles in various cellular activities, deregulation of proteinkinase enzymatic activity can lead to altered cellular properties, suchas uncontrolled cell growth associated with cancer. In addition tooncological indications, altered kinase signaling is implicated innumerous other pathological diseases, including, for example,immunological disorders, cardiovascular diseases, inflammatory diseases,and degenerative diseases. Therefore, protein kinases are attractivetargets for small molecule drug discovery. Particularly attractivetargets for small-molecule modulation with respect to antiangiogenic andantiproliferative activity include receptor type tyrosine kinases Ret,c-Met, and VEGFR2.

The kinase c-Met is the prototypic member of a subfamily ofheterodimeric receptor tyrosine kinases (RTKs) which include Met, Ron,and Sea. The endogenous ligand for c-Met is the hepatocyte growth factor(HGF), a potent inducer of angiogenesis. Binding of HGF to c-Met inducesactivation of the receptor via autophosphorylation resulting in anincrease of receptor dependent signaling, which promotes cell growth andinvasion. Anti-HGF antibodies or HGF antagonists have been shown toinhibit tumor metastasis in vivo (See Maulik et al, Cytokine & GrowthFactor Reviews, 2002, 13, 41-59). c-Met, VEGFR2, and/or Retoverexpression has been demonstrated on a wide variety of tumor types,including breast, colon, renal, lung, squamous cell myeloid leukemia,hemangiomas, melanomas, and astrocytic tumor (which includesglioblastoma, giant cell glioblastoma, gliosarcoma, and glioblastomawith oligodendroglial components). The Ret protein is a transmembranereceptor with tyrosine kinase activity. Ret is mutated in most familialforms of medullary thyroid cancer. These mutations activate the kinasefunction of Ret and convert it into an oncogenic form.

Accordingly, small-molecule compounds that specifically inhibit,regulate, and/or modulate the signal transduction of kinases,particularly including Ret, c-Met, and VEGFR2 described above, areparticularly desirable as a means to treat or prevent disease statesassociated with abnormal cell proliferation and angiogenesis. One suchsmall-molecule is XL184, known variously asN-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamideand by the name cabozantinib (COMETRIQ™), which is the L-malate salt ofcabozantinib. Cabozantinib has the chemical structure:

In November, 2012, cabozantinib achieved regulatory approval in theUnited States for the treatment of progressive metastatic medullarythyroid cancer. Other clinical trials of cabozantinib are ongoing.

WO 2005/030140 describes the synthesis of cabozantinib (Example 48) andalso discloses the therapeutic activity of this molecule to inhibit,regulate, and/or modulate the signal transduction of kinases, (Assays,Table 4, entry 289). Example 48 is on paragraph [0353] in WO2005/030140.

A need remains for identifying compounds that exhibit a similar activityprofile to cabozantinib.

SUMMARY OF THE INVENTION

These and other needs are met by the present invention, which isdirected to metabolites of cabozantinib.

In one embodiment of this aspect, the metabolite is a compound offormula Ia

having one or more of the following attributes:

a) one of R₁ or R₂ is H, SO₃H, or a glucuronic acid moiety, and theother is Me;

b) R₃ is OH or OSO₃H;

c) R₄ is O⁻, provided that when R₄ is O⁻, N is N⁺;

d) R₅ is OH, or OSO₃H; and

e) R₆ is OH or OSO₃H.

In another embodiment of this aspect, the metabolite is a compound offormula Ib

wherein:

-   -   a) R₁ or R₂ are Me; or one of R₁ or R₂ is H, SO₃H, or a        glucuronic acid moiety, and the other is Me;    -   b) R₃ is H, OH, or OSO₃H;    -   c) R₄ is absent or is O⁻, provided that when R₄ is O⁻, N is N⁴;        and    -   d) R₆ is H or Me.

In another embodiment of this aspect, the metabolite is a compound offormula Ic

wherein:

-   -   a) R₅ is OH or OSO₃H; and    -   b) R₆ is OH or OSO₃H; and    -   c) R₇ is H, SO₃H, or a glucuronic acid moiety.

In one aspect, the invention is directed to an isolated metabolite ofcabozantinib having formula Ia, Ib, or Ic.

In one embodiment, the metabolite of cabozantinib is selected from:

wherein GA is a glucuronic acid moiety such as in, for example,

In another aspect, the invention is directed to a compound which isselected from:

wherein GA is a glucuronic acid moiety.

In another aspect, the invention is directed to a method of treatingdiseases or disorders associated with uncontrolled, abnormal, and/orunwanted cellular activities, the method comprising administering, to amammal in need thereof, a therapeutically effective amount of a compoundwhich is a metabolite of cabozantinib. In one embodiment, the metaboliteis selected from:

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention is directed to a pharmaceuticalcomposition comprising a therapeutically active metabolite ofcabozantinib and at least one pharmaceutically acceptable carrier. Inone embodiment, the metabolite is selected from:

or a pharmaceutically acceptable salt thereof and at least onepharmaceutically acceptable carrier.

In another aspect the invention is directed to a method for identifyinga metabolite of cabozantinib, comprising:

administering cabozantinib to a mammal;

detecting or measuring a level or concentration of a metabolite ofcabozantinib in a mammal in tissues or bodily fluids of the mammal;

wherein the metabolite is selected from the group consisting of:

wherein GA is a glucuronic acid moiety.

The compounds may additionally be used in other methods; for example, inbioassay methods for determining the kinase inhibitory activity of testcompounds or as internal standards in related methods.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention is directed to metabolites of cabozantinib,particularly human metabolites. Thus, the metabolites may be referred tohereinafter as “human metabolites.” Human metabolites of cabozantinibinclude metabolites of cabozantinib that were formed in the bodies ofhuman subjects after ingestion or application of cabozantinib accordingto clinical protocols regarding dosing and monitoring, including thosedescribed herein. In various embodiments, the term encompasses molecularspecies formed in vivo, whether or not the species is even detected oranalyzed in a particular trial. It is also contemplated that somemetabolites are unique to particular individuals, reflecting differentgenetic make-up and the presence and activity of various enzymes,including cytochrome P450 and UGT enzymes which are involved inmetabolism. Thus, human metabolites cover all such metabolites formed inthe human body.

Some human metabolites are depicted in Scheme 1. These human metaboliteswere identified during clinical studies of cabozantinib, which appearsas compound I in Scheme 1, by metabolic profiling, particularly fromhuman plasma, urine, and feces.

In various embodiments, the cabozantinib metabolites, including thosedepicted in Scheme 1, are isolated from body tissues and fluids, and/orare prepared synthetically according to methods available to the skilledartisan. A variety of separation processes can be carried out on tissueand fluid samples to provide samples for further analysis, such asnuclear magnetic resonance, gas chromatography (GC), liquidchromatography (LC), and mass spectrometry. In such samples, themetabolites are contained in compositions that are essentially lackingin the presence of any of the other metabolites. The presence of themetabolites can be quantified by physical methods, such as themeasurement of nuclear decay from radioactive isotopes, measurement ofindex of refraction, flame ionization, ionization and deflection inmagnetic fields, ultraviolet (UV absorption), and the like.

In various embodiments, the human metabolites are provided incrystalline or solution forms that have considerable degrees of purity.Organic synthetic routes are available for preparing the compounds inrelative pure form, for example, in purifies of 80 percent or greater,90 percent or greater, 95 percent or greater, or 99 percent or greater.Recrystallization and other purification methods can be carried out toprovide compounds that are essentially 100 percent pure. Such syntheticmethods and purification techniques are known in the art.

In various embodiments, the metabolites are provided in substantiallypure form. “Substantially pure” means that metabolites are pure enoughfor FDA approval and contain essentially no contaminants or othermaterials. Alternatively, “substantially pure” means a level of impuritythat does not adversely or unacceptably affect the properties of thecompounds with respect to safety, effectiveness, stability, and otherdesirable properties.

In one embodiment, the invention is directed to isolated metabolites asdepicted in Scheme 1. In this and other embodiments, the metabolite isselected from:

wherein GA is a glucuronic acid moiety.

More particularly, the metabolite is selected from:

In a particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the isolated metabolite is

or a pharmaceutically acceptable salt thereof.

Methods of the invention include administering cabozantinib or acabozantinib metabolite to a mammal such as a human and detectingmetabolites by measuring the level of concentration of one of themetabolites in in the tissues or bodily fluids of the mammal. Bodilyfluids include, without limitation, blood plasma, bile, urine, andfeces, while tissues include, without limitation, liver microsomes,hepatocytes, and perfused livers. In various embodiments, themetabolites are isotopically labeled with various isotopes to assist indetection or quantification of the metabolites in in the tissues orbodily fluids. Thus, the metabolites include those that are labeled with¹⁴C or ³H (tritium) for the purpose of detecting or identifyingparticular metabolites from their nuclear decay products. Themetabolites also include metabolites labeled with ¹³C or ²H (deuterium)to facilitate nuclear magnetic resonance and/or mass spectrometricanalysis of the compounds. As used herein, deuterated means substitutedwith deuterium, and tritiated means substituted with tritium. In variousother embodiments, the metabolites of the invention, as depicted inScheme 1, also include their salts, tautomers, and isotopically labeledvariants (including ¹⁴C, ¹³C, ³H, or ²H).

More specifically, in one embodiment, the invention provides a methodfor identifying a metabolite of cabozantinib, comprising:

administering cabozantinib to a mammal;

detecting or measuring a level or concentration of a metabolite ofcabozantinib in a mammal in tissues or bodily fluids of the mammal;

wherein the metabolite is selected from the group consisting of:

wherein GA is a glucuronic acid moiety. In the method, bodily fluids areselected from the group consisting of plasma, bile, urine, and feces. Inthese and other methods, the metabolites are identified usingconventional analytical techniques, including isotopic labeling andHPLC/MS.

More specifically, the metabolite is selected from:

Another aspect of the invention is a method of modulating the in vivoactivity of a kinase, the method comprising administering to a subjectan effective amount a cabozantinib metabolite, which is a compoundselected from:

or a pharmaceutical composition comprising such a compound.

In one embodiment of this aspect, modulating the in vivo activity of thekinase comprises inhibition of said kinase.

In another embodiment of this aspect, the kinase is at least one ofc-Met, RET, KDR, c-Kit, flt-3, and flt-4.

In another embodiment, the kinase is c-Met.

Another aspect of the invention is directed to a method of treatingdiseases or disorders associated with uncontrolled, abnormal, and/orunwanted cellular activities, the method comprising administering, to amammal in need thereof, a therapeutically effective amount of acabozantinib metabolite, which is a compound selected from:

or a pharmaceutical composition comprising such a compound.

In a particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention is directed to a method of screeningfor a modulator of a kinase, said kinase selected from c-Met, KDR, RET,c-Kit, flt-3, and flt-4, the method comprising combining a cabozantinibmetabolite which is a continued selected from:

and at least one candidate agent, and determining the effect of thecandidate agent on the activity of said kinase.

Another aspect of the invention is directed to a method of inhibitingproliferative activity in a cell, the method comprising administering aneffective amount of a composition comprising a compound to a cell or aplurality of cells, wherein the compound is a cabozantinib metaboliteselected from:

Another aspect of the invention is a method of screening for a modulatorof a kinase, said kinase selected from c-Met, KDR, RET, c-Kit, flt-3,and flt-4, the method comprising combining a compound and at least onecandidate agent and determining the effect of the candidate agent on theactivity of said kinase, wherein the compound is a cabozantinibmetabolite selected from:

Isolated metabolites as described above that exhibit inhibitory activityagainst c-MET or other kinases can be formulated into suitable dosageforms for administration to humans or other mammals. In someembodiments, the metabolites may exhibit favorable toxicologicalprofiles in comparison to conventional therapy or therapy withcabozantinib.

As inhibitors of c-MET, in some embodiments, the metabolites are used totreat cancer. “Cancer” includes tumor types such as tumor typesincluding breast, colon, renal, lung, squamous cell myeloid leukemia,hemangiomas, melanomas, astrocytomas, and glioblastomas as well as othercellular-proliferative disease states, including but not limited to:Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:bronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushanlartoma, inesothelioma; Gastrointestinal: esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,insulinorna, glucagonoma, gastrinoma, carcinoid tumors, vipoma), smallbowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia, renal cell carcinoma), bladder andurethra (squamous cell carcinoma, transitional cell carcinoma,adenocarcinoma), prostate (adenocarcinoma, sarcoma, small cell carcinomaof the prostate), testis (seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma,fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), malignant giant cell tumor chordoma, osteochronfroma(osteocartilaginous exostoses), benign chondroma, chondroblastoma,chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervoussystem: skull (osteoma, hemangioma, granuloma, xanthoma, osteitisdefornians), meninges (meningioma, meningiosarcoma, gliomatosis), brain(astrocytoma, medulloblastoma, glioma, ependymoma, germinoma[pinealoma], glioblastorna multiform, oligodendroglioma, schwannoma,retinoblastoma, congenital tumors), spinal cord neurofibroma,meningioma, glioma, sarcoma); Gynecological: uterus (endometrialcarcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia),ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinouscystadenocarcinoma, unclassified carcinoma], granulosa-thecal celltumors, Sertoli Leydig cell tumors, dysgerminoma, malignant teratoma),vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma],fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma;as well as cancers of the thyroid including medullary thyroid cancer.Thus, the term “cancerous cell,” as provided herein, includes a cellafflicted by any one of the above-identified conditions.

In one embodiment, the cancer is selected from ovarian cancer, prostatecancer, lung cancer, medullary thyroid cancer, liver cancer,gastrointestinal cancer, pancreatic cancer, bone cancer, hematologiccancer, skin cancer, kidney cancer, breast cancer, colon cancer, andfallopian tube cancer.

In another embodiment, the disease or disorder is ovarian cancer.

In another embodiment, the disease or disorder is prostate cancer.

In another embodiment, the disease or disorder is lung cancer.

In another embodiment, the disease or disorder is medullary thyroidcancer.

In another embodiment, the disease or disorder is liver cancer.

In another embodiment, the disease or disorder is gastrointestinalcancer.

In another embodiment, the disease or disorder is pancreatic cancer.

In another embodiment, the disease or disorder is bone cancer.

In another embodiment, the disease or disorder is hematologic cancer.

In another embodiment, the disease or disorder is skin cancer.

In another embodiment, the disease or disorder is kidney cancer.

In another embodiment, the disease or disorder is breast cancer.

In another embodiment, the disease or disorder is colon cancer.

In another embodiment, the disease or disorder is fallopian cancer.

In another embodiment, the disease or disorder is liver cancer, whereinthe liver cancer is hepatocellular carcinoma, cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, or hemagioma.

In another embodiment, the disease or disorder is gastrointestinalcancer, wherein the gastrointestinal cancer is cancer of the esophagouswhich is squamous cell carcinoma, adenocarcinoma, or leiomyosarcoma;cancer of the stomach which is carcinoma, or lymphoma; cancer of thepancreas, which is ductal adenocarcinoma, insulinoma, gucagonoma,gastrinoma, carcinoid tumors, or vipoma; cancer of the small bowel,which is adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemagioma, lipoma, or cancer of the large bowel, which isadenocarcinoma, tubular adenoma, villous adenoma, hamartoma, orleiomyoma.

In another embodiment, the disease or disorder is cancer of thepancreas, wherein the cancer of the pancreas is ductal adenocarcinoma,insulinoma, gucagonoma, gastrinoma, carcinoid tumors, or vipoma.

In another embodiment, the disease or disorder is bone cancer, whereinthe bone cancer is osteosarcoma, fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant reticulum cellsarcoma, multiple myeloma, malignant giant cell tumor chordoma,osteocartiliginous exostoses, chondroblastoma, chondromyxofibroma, orosteoid osteoma.

In another embodiment, the disease or disorder is hematologic cancer,wherein the hematologic cancer is myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, or myelodysplastic syndrome.

In another embodiment, the disease or disorder is skin cancer, whereinthe skin cancer is malignant melanoma, basal cell carcinoma, squamouscell carcinoma, or Karposi's sarcoma.

In another embodiment, the disease or disorder is a renal tumor or renalcell carcinoma.

In another embodiment, the disease or disorder is breast cancer.

In another embodiment, the disease or disorder is a colon cancer tumor.

In another embodiment, the disease or disorder is fallopian tubecarcinoma.

Alternatively, or additionally, the metabolites are administered tosubjects or test animals not having any of the above mentioned diseasestates for the purpose of studying non-pharmacological effects, such asside effects, toxicity, metabolism, uptake, bioavailability, and routesof excretion.

In various embodiments, the metabolites are administered by any suitableroute including oral, rectal, intranasal, intrapulmonary (e.g., byinhalation), or parenteral (e.g. intradermal, transdermal, subcutaneous,intramuscular, or intravenous) routes. Oral administration is preferredin some embodiments, and the dosage can be given with or without food,i.e. in the fasting or non-fasting state. Non-limiting examples ofdosage forms include uncoated or coated tablets, capsules, powders,granules, suppositories, solutions, ointments, creams, and sprays.

Formulations of the invention suitable for oral administration areprepared as discrete units, such as capsules, cachets, or tablets, eachcontaining a predetermined amount of the active ingredient; as a powderor granules; as solution or a suspension in an aqueous liquid or anon-aqueous liquid; or as an oil-in-water liquid emulsion; or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary, or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form, suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active, or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom. In one embodiment, acid hydrolysis of the medicament isobviated by use of an enteric coating.

An enteric coating is a means of protecting a compound of the inventionin order to avoid exposing a portion of the gastrointestinal tract,typically the upper gastrointestinal tract, in particular the stomachand esophagus, to the compound of this invention. In this way, gastricmucosal tissue is protected against rates of exposure to a compound ofthe invention which produce adverse effects such as nausea; and,alternatively, a compound of the invention is protected from conditionspresent in one or more portions of the gastrointestinal tract, typicallythe upper gastrointestinal tract.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

While it is possible for the active ingredients to be administeredalone, it may be preferable to present them as pharmaceuticalformulations. The formulations, both for veterinary and for human use,comprise at least one active ingredient, as defined above, together withone or more acceptable carriers and optionally comprising othertherapeutic ingredients. The carrier(s) must be “acceptable” in thatthey are compatible with the other ingredients of the formulation andphysiologically innocuous to the recipient thereof.

In various embodiments the compounds are formulated in a carrier system.Such systems are known and include binders, fillers, preservatives,disintegrants, flow regulators, plasticizers, wetting agents,emulsifiers, dispersants, lubricants, solvents, release slowing agents(including enteric coatings), antioxidants, and propellant gases.Especially when formulated for administration to humans, the activeagents are preferably combined with at least one pharmaceuticallyacceptable carrier. Such carriers are known and include, withoutlimitation, cellulose derivatives, polyethylene glycol, andN-vinylpyrrolidone polymers. The administration forms comprise atherapeutically effective amount of the compounds, which make up from0.1% to about 90% by weight of the dosage form.

The compounds of this invention are formulated with conventionalcarriers and excipients, which are selected in accord with ordinarypractice. Tablets will contain excipients, glidants, fillers, binders,and the like. Aqueous formulations are prepared in sterile form and,when intended for delivery by other than oral administration, generallywill be isotonic. All formulations will optionally contain excipients,such as those set forth in the “Handbook of Pharmaceutical Excipients”(1986). Excipients include ascorbic acid and other antioxidants,chelating agents such as EDTA, carbohydrates such as dextrin,hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, andthe like.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

In a particular embodiment, the invention provides a pharmaceuticalcomposition comprising a cabozantinib metabolite which is a compoundselected from:

or a pharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable carrier.

The compounds disclosed herein can be made according to methodsavailable to the skilled practitioner. For example, as depicted inScheme 2, peptide chemistry can be used to make the phenols C-1 and C-2from the corresponding amines and carboxylic acids. A variety ofprocesses and reagents are available for achieving such transformationsand are described, for instance, in Tetrahedron 61 (2005) 10827-10852. Arepresentative example is depicted in Scheme 2, wherein the activatingagent is thionyl chloride, oxalyl chloride, or the like. Thecorresponding acid chloride reacts with compound A or B, respectively,to provide phenol C-1 or C-2. Subsequent reaction of phenol C-1 or C-2with a sulfating agent, such as chlorosulfonic acid or sulfurtrioxide-trimethylamine complex, in the presence of a base, such astriethylamine, alkali metal hydroxide or the like, can provide thecorresponding hydrogen sulfate 2b or 2a, respectively.

Compound A was prepared according to Scheme 3. Benzylation of A-1 usinga benzyl halide or the like provides benzyl-protected A-2. Nitration ofA-2 using a mixture of nitric acid and sulfuric acid provides A-3.Reduction of nitro group in A-3 to the amine A-4, may be accomplishedusing standard nitro reduction conditions, such as iron and ammoniumacetate. Cyclization of A-4 with ethyl formate and an alkoxide such assodium methoxide provides the A-5. Conversion of A-5 to thecorresponding chloride using phosphorous oxychloride provides A-6.Reaction of A-6 with 4-amino phenol provides A-7, which is deprotectedwith methane sulfonic acid to provide compound A.

Similarly, compound B was prepared according to Scheme 4. Demethylationof B-1 provides B-2. Benzylation of B-2 using a benzyl halide BnX,wherein X is Br Cl or I, or the like provides benzyl-protected B-3.Nitration of B-3 using a mixture of nitric acid and sulfuric acidprovides B-4. Reduction of nitro group in B-4 to the amine B-5, may beaccomplished using standard nitro reduction conditions, such as iron andammonium acetate. Cyclization of B-5 with ethyl formate and an alkoxidesuch as sodium methoxide provides the B-6. Conversion of B-6 to thecorresponding chloride using phosphorous oxychloride provides B-7.Reaction of B-7 with 4-amino phenol provides B-8, which was deprotectedwith methane sulfonic acid to provide compound B.

Phenols 13 and 16 can be similarly prepared from compound 7, thesynthesis of which is disclosed in WO 2005/030140 as Example 73. Thus,in Scheme 5, coupling of 7 with 2-amino-5-fluorophenol (CAS Reg. No.53981-24-1) provides 13. Coupling of 7 with 5-amino-2-fluorophenol (CASReg. No. 100367-48-4) provides 16.

Phenols 13 and 16 can be readily converted to the corresponding sulfates9, and 12 depicted in Scheme 1 using, for example, a sulfating agent,such as sulfur trioxide trimethyl amine complex, in the presence of astrong hydroxide, such as potassium hydroxide, sodium hydroxide, or thelike, or using chlorosulfonic acid in the presence of an amine base suchas triethylamine.

The phenols 15a and 15b can be prepared by employing the similar methodthat is disclosed in WO 2005/030140 for the preparation of Example 43.Thus, in Scheme 6, coupling of phenol C (Example 38 in WO 2005/030140)with triflate D (Example 33 in WO 2005/030140), or chloride A-6 (Example32 in WO 2005/030140) provides E, which is then deprotected underPd-catalyzed hydrogenolysis condition to yield compound 15 Similarly,reaction of phenol C with triflate F or chloride B-7 provides G, whichis subjected to O-benzyl deportection to provide compound 15b.

The N-oxide 19 can be prepared by the reaction of cabozantinib with anoxidizing agent, such as, for example a peroxide, a peracid, or thelike. In one embodiment, the oxidizing agent is sodium perboratetetrahydrate.

The following non-limiting examples are meant to illustrate theinvention.

Examples Identification of Cabozantinib Metabolites

The objective of this study was to profile and identify metabolites ofcabozantinib in human plasma, urine, and feces. The plasma, urine andfecal samples were collected from a mass balance study of cabozantinibfollowing a single 175 mg oral administration of cabozantinib (L-malatesalt) containing [¹⁴C] cabozantinib (100 μCi) in healthy male subjects.

Study Design and Plasma, Urine, and Feces Sampling

Eight male subjects participated in the study, and each subject receiveda single oral dose of 175 mg of cabozantinib (L-malate salt) containing[¹⁴C]-cabozantinib (100 μCi). The plasma, urine, and fecal samples werecollected from the 8 subjects for the metabolite profiling.

Plasma samples were collected at pre-dose, 0.5, 1, 2, 3, 4, 5, 8, 14,24, 72, 168, 336, 504 and 648 hours post-dose; urine samples werecollected at pre-dose, 0-8 hours, 8-24 hours, at 24-hour intervals to480 hours post-dose, and at greater than 48-hour intervals from 504 to1152 hours post-dose; and feces samples were collected at pre-dose, at24-hour intervals to 480 hours post-dose, and at greater than 48-hourintervals from 504 to 1152 hours post-dose. All samples were shipped toQPS LLC (Newark, Del.) and stored at −70° C. HPLC/tandem MS coupled witha radio flow-through detector (RFD) was used for metabolite profilingand identification for samples with sufficient levels of radioactivity.

HPLC fraction collection followed by counting with TopCount NXT™ wasused for radioquantitation of plasma samples with sufficient levels ofradioactivity. Three (3) HPLC methods were used in this study toseparate cabozantinib and its metabolites. HPLC Method l was used forthe analysis of pooled urine and fecal samples and individual plasmasamples from different time points. HPLC Method 2 was used for theanalysis of plasma samples from a drug-drug interaction study to searchfor possible metabolites that may co-elute with cabozantinib sulfate.HPLC Method 3 was used for pooled plasma samples.

Selected samples for plasma, urine, and feces from 6 subjects wereanalyzed for cabozantinib and metabolites and reported.

Samples from 2 subjects were used for the investigation study.

Test Article

The test article for this study was a mixture of [¹⁴C] cabozantinib andcabozantinib. The asterisk indicates the position of the [¹⁴C] label.[¹⁴C] labeled cabozantinib was prepared as provided in WO 2005/030140,except that [¹⁴C] labeled 4-amino phenol was used instead of unlabeled4-amino phenol. [¹⁴C] labeled 4-amino phenol is commercially availableas the hydrochloride salt, for instance, from Hartmann Analytic,American Radiolabeled Chemicals, or Fisher Scientific.

General Chemicals and Reference Standards

Formic acid and ammonium acetate were obtained from Sigma-AldrichChemical Co. (St. Louis, Mo.). Acetonitrile (B & J brand, carbonyl free,for applications sensitive to dace aldehyde and ketone), water (B & Jbrand, for GC, HPLC and spectrophotometry), and methanol (HPLC grade)were purchased from Fisher Scientific (Pittsburgh, Pa.). Type I waterwas generated using an Elgastat UHQ PS water purification system.Non-radiolabeled metabolite standards (fluoroaniline cleavage product,cabozantinib sulfate, and cabozantinib N-oxide) were provided byExelixis, Inc.

Biological Sample Collection

The plasma, urine, and fecal samples were collected from a mass balancestudy of cabozantinib following a single 175 mg oral administration ofcabozantinib (L-malate salt) containing [¹⁴C] cabozantinib (100 μCi) inhealthy male subjects. Samples were shipped from Celerion (Lincoln,Nebr.) to QPS LLC (Newark, Del.) on dry ice and were stored at −70° C.until analysis. Samples from 6 subjects were used for metaboliteprofiling, identification, and radio-quantitation. Plasma samples from 2subjects were only used in a bridging study as part of investigation ofco-eluting metabolites.

Sample Preparation and Radioactive Recovery for Human Plasma

For metabolite profiling, identification, and radio-quantitation,individual radiolabeled plasma samples collected at 0.5, 1, 2, 3, 4, 5,8, 14, 24, 72, 168, and 336 hours post-dose were processed and analyzedfor 6 subjects. For the investigation of co-eluting metabolites,nonradiolabeled plasma samples of six subjects were pooled, processed,and analyzed for pre-dose, 1-7, 8-96, and 120-336 hours post-dose. Tobridge the metabolite data from non-radiolabeled to radiolabeled plasmasamples from the human mass balance study, [¹⁴C] plasma samples from0-168 hours post-dose for each of the six subjects were also pooledusing the Hamilton pooling method, processed, and analyzed byradio-quantitation. [¹⁴C] Plasma samples from 1-168 hours post-dose fortwo subjects were pooled (equal volume), processed, and analyzed.

Initial Method for Plasma Extraction and Recovery

Two plasma samples from a subject (4 and 72 hours post-dose) were usedfor initial extraction and recovery determination. The totalradioactivity for each plasma sample in mass balance study was providedby Exelixis, Inc., and was defined as 100%. After the samples werethawed under a biological hood, two 0.5 mL aliquots of each plasmasample were added to 3 volumes (1.5 mL) of MeOH:ACN (20:80, v/v) andvortexed (5 min). The mixtures were centrifuged at 2000 rpm for 10minutes, and the supernatants were transferred to clean tubes. Thepellets were extracted with two additional 3 volumes of MeOH:CAN (20:80,v/v). The mixtures were centrifuged, and the supernatants were combined.Aliquots were analyzed by a 2900 TR liquid scintillation counter (LSC)(Packard Instruments, Meridian, Conn.). The extraction recovery wascalculated as the following:

Extraction Recovery (%)=(DPM in supernatant/DPM in plasma sample)×100

The supernatants from the extraction were evaporated to dryness under astream of nitrogen in an ambient water bath. The residues were thenreconstituted in 0.35-0.5 mL of MeOH:ACN:water (10:20:70, v/v/v). Thereconstituted samples were centrifuged at 15,000 rpm for 10 minutes andaliquots were analyzed by LSC for reconstitution recovery.

Reconstitution Recovery (%)=(DPM in reconstitution solution/DPM insupernatant)×100

Plasma Sample Preparation

Radiolabeled and non-radiolabeled plasma samples were extractedemploying the same method, using 1.0-2 mL plasma samples, depending onthe volume available and radioactivity level of the samples. Thesupernatants were evaporated to dryness under a stream of nitrogen in anambient water bath, and the residues were reconstituted in 0.35-0.5 mLof MeOH:ACN:water (10:20:70, v/v/v). The reconstituted samples werecentrifuged at 15,000 rpm for 10 minutes. Aliquots of the supernatantswere injected onto the HPLC system for analysis.

Sample Preparation and Radioactive Recovery for Human Urine

Pooled urine samples from a subject (0-72, 168-192, and 312-336 hourspost dose) were lyophilized in triplicate (each 4 mL), and the residueswere reconstituted in 1 mL of water:ACN:FA (80:20:0.1, v/v/v). Theradioactivity in pooled urine and reconstituted solution was countedusing LSC, and the reconstitution recovery calculated. For metaboliteprofiling, identification, and radio-quantitation, pre-dose and 3 pooledurine samples (0-72 hours, 168-192 hours, and 312-336 hours post-dose)from each of the six subjects were analyzed. Each pooled urine samplewas lyophilized, the residue was reconstituted in water:ACN:FA(80:20:0.1, v/v/v), and the reconstituted sample was centrifuged at15,000 rpm for 10 min before analysis.

Sample Preparation and Radioactive Recovery for Human Urine

To evaluate the extraction recovery of fecal samples, two fecalhomogenate samples from a subject were thawed under a biological hood.Approximately 5.5-6 g fecal homogenate was accurately weighed out forthe extraction. Fifteen mL ACN:MeOH (80:20) was added to the fecalhomogenates. The mixtures were vortexed for 3 minutes and centrifuged at3000 rpm for 10 minutes. The supernatants were transferred to cleantubes. The extraction procedure was repeated two more times. Thesupernatants from all three extractions were combined. The radioactivityin the combined supernatants was determined by LSC. The extractionrecovery was calculated using the following formula:

Extraction Recovery (%)=(DPM in supernatant/DPM in fecal homogenate)×100

The supernatant was concentrated under a nitrogen stream at ambienttemperature, and the residues were reconstituted in MeOH:ACN:water(10:20:70). Aliquots of reconstitution solution were counted with LSCfor reconstitution recovery.

Reconstitution Recovery (%)=(DPM in reconstitution solution/DPM insupernatant)×100

Overall Recovery (%)=Extraction Recovery (%)×Reconstitution Recovery(%)/100

For metabolite profiling, identification, and radio-quantitation,pre-dose and 3 pooled fecal samples (0-72, 168-192, and 312-336 hourspost-dose) from each of the six subjects were extracted using the sameprocedures for extraction recovery. The supernatants were dried under anitrogen stream, and the residues were reconstituted in water:ACN:FA(80:20:0.1, v/v/v). The reconstituted samples were centrifuged at 15,000rpm for 10 min before analysis.

HPLC Column Recovery

HPLC column recovery was carried out to demonstrate that all radioactivecomponents were effectively eluted from the column using HPLC Method 1.Aliquots of urine samples (Subject 1042, 24-48 hours post-dose) wereinjected onto the HPLC system with or without a column, and the eluentsfrom 0-30 minutes were collected into clean 50 mL centrifuge tubes. Theweights of eluent from each injection were obtained after collection,and duplicate aliquots (1 mL) were counted using LSC. The average valueof the counts was used to calculate the total radioactivity contained inthe collected eluent with or without a column installed.

Column Recovery (%)=(DPM in eluent with column/DPM in eluent withoutcolumn)×100

HPLC Method 3 was used for pooled plasma only, and the column recoverywas not performed due to limited sample volume available.

HPLC/MS/RFD and HPLC Radio-Analysis Systems

The system for metabolite profiling and identification (HPLC/MS/RFD)consisted of a HTC PAL autosampler, a Surveyor HPLC pump, a LTQ linearion trap mass spectrometer, and a β-RAM Model 3 RFD. The data obtainedby mass spectrometry and RFD were processed by Xcalibur and Laura Lite 3software, respectively. The HPLC eluent was split between the RFD andmass spectrometer with a ratio of 3 to I. The following are the summaryof the conditions for HPLC, mass spectrometer, and RFD.

HPLC/MS/RFD Method 1

HPLC Surveyor HPLC pump Column Type Phenominex Synergi Polar RP, 4.6 ×250 mm, 4 μm Mobile Phases A: H₂O with 0.1% FA B: ACN with 0.1% FAGradient Program Time (min) A % B % 0 80 20 2 80 20 22 30 70 23 5 95 275 95 28 80 20 34 80 20 Flow Rate 800 μL/minutes Analysis Time 34 minutesMass Spectrometry: Thermo Finnigan LTQ Linear Ion Trap Sheath gas flowrate 50 unit Auxiliary gas flow rate 20 unit Sweep gas flow rate 10 unitIon spray voltage 5 kV for ESI+; 4.3 kV for ESI− Capillary temperature300° C. Capillary voltage 22 V for ESI+: −9 V for ESI− Tube lens voltage80 V for ESI+: −96 V for ESI− Ionization mode ESI−, ESI− RadioFlow-through Detector: β-RAM Model 3 Radionuclide ¹⁴C Cell Volume 400 μLScintillation Cocktail Ultima-Flo M, Perkin Elmer Cocktail/HPLC flowratio 3:1

HPLC/MS Method 2

HPLC Surveyor HPLC pump Column Type Phenominex Synergi Polar RP, 4.6 ×250 mm, 4 μm Mobile Phases A: H₂O with 0.1% FA B: ACN with 0.1% FAGradient Program Time (min) A % B % 0 80 20 2 80 20 40 35 65 42 5 95 475 95 48 80 20 55 80 20 Flow Rate 800 μL/minutes Analysis Time 55 minutesMass Spectrometry: Thermo Finnigan LTQ Linear Ion Trap Sheath gas flowrate 50 unit Auxiliary gas flow rate 20 unit Sweep gas flow rate 10 unitIon spray voltage 5 kV Capillary temperature 300° C. Capillary voltage22 V Tube lens voltage 80 V Ionization mode ESI+

HPLC/MS Method 3

HPLC Surveyor HPLC pump Column Type Waters Xbridge phenyl, 4.6 × 150 mm,3.5 μm Mobile Phases A: H₂O with 0.1% FA B: ACN with 0.15% FA GradientProgram Time (min) A % B % 0 80 20 2 80 20 40 30 70 42 5 95 47 5 95 4880 20 55 80 20 Flow Rate 800 μL/minutes Analysis Time 55 minutes MassSpectrometry: Thermo Finnigan LTQ Linear Ion Trap Sheath gas flow rate50 unit Auxiliary gas flow rate 20 unit Sweep gas flow rate 10 unit Ionspray voltage 5 kV Capillary temperature 300° C. Capillary voltage 22 VTube lens voltage 80 V Ionization mode ESI+

The HPLC-MS system for high resolution MS consisted of a MichromBioresources Paradigm MS4B HPLC and a Thermo LTQ Orbitrap Discovery massspectrometer. Chromatographic conditions and the ion source parameterswere the same as HPLC method 1 for the LTQ system. Data were acquiredwith a resolution of 30000 in centroid mode.

An HPLC/TopCount NXT™ system was used for the radio-quantitation ofplasma samples. The system consisted of an HTC PAL autosampler, twoShimadzu HPLC pumps, and a Foxy Jr. Fraction Collector (Isco, Lincoln,Nebr.). HPLC fractions collected in a LumaPlate™ 96-well plate weredried using an EZ-2_(plus) Personal Evaporator (Genevac, Valley Cottage,N.Y.), and the dried samples were counted by TopCount NXT™ MicroplateScintillation & Luminescence Counter (PerkinElmer®). The data wereprocessed using ProFSA (PerkinElmer®) software. The HPLC methods werethe same as described above.

Metabolite Identification

Metabolites that represented greater than 5% of the total radioactivityor 5% of total AUC in the matrix were identified according to thefollowing process. Mass spectra (MS, MS/MS, and MS/MS/MS) ofcabozantinib and its metabolite standards, provided by the Exdlixis,Inc., were acquired on an ion trap mass spectrometer. Major fragmentpatterns were proposed. Identification of these metabolites wasconfirmed by matching mass spectra (MS and MS/MS) and retention timeswith authentic reference standards. For other unknown metabolites,molecular ions were searched on LC/MS chromatograms operating in fullscan positive as well as negative ionization modes at the same retentiontimes as those found on LC-radio chromatogram. Product ion mass spectraand high resolution mass spectra were then acquired for thecorresponding molecular ions. Putative metabolite structures wereproposed based on the analysis of their mass fragment patterns.

Quantitation of Cabozantinib and its Metabolites

Quantitation of cabozantinib and its metabolites in pooled or individualsamples from each matrix at different time points or time intervals wasbased on integration of the corresponding peaks found on theirradio-chromatograms. For plasma samples, percent of total radioactivityin the sample for each peak at each time point was calculated andconverted to ng/mL.

For quantification of cabozantinib and its metabolites in plasma:

ng/mL=(% of the total radioactivity)×(total ng equivalent/mL for thetime point)/100

The values of total ng equivalent/mL were obtained from the results ofthe human mass balance study.

For the pooled urine samples, percent of total radioactivity in thepooled sample for each peak was calculated as the % of total non-parentin the pooled samples:

% of total non-parent in the pooled samples=(total radioactivity of thepeak/total radioactivity of the non-parent peaks)×100

For the pooled fecal samples, percent of total radioactivity in thepooled sample for each peak was calculated as the percent of totalnon-parent plus parent in the pooled samples:

% of total non-parent plus parent in the pooled samples=(totalradioactivity of the peak/total radioactivity of the parent andnon-parent peaks)×100

The percent of total radioactivity in the pooled sample for each peakwas converted to the percent of parent in the pooled samples:

% of parent in the pooled samples=(total radioactivity of the peak/totalradioactivity of the parent peak)×100

The limit of quantification for a radioactivity detector was defined asthe ratio of signal to noise (3 to 1) on the radio-chromatogram. The lowlimits of quantification were 10 and 500 dpm for the TopCount and a-RAMradio flow-through detector, respectively.

Results and Discussion

Radioactive Recovery

The initial extraction recovery was determined using plasma samples froma subject at 4 hours and 72 hours post-dose with three volumes ofMeOH:ACN (20:80) extracting three times. The mean extraction recoveriesof radioactivity from 4 and 72 hour samples were 98.43% and 94.99%,respectively. After drying down and reconstitution into MeOH:ACNsolution, the reconstitution recoveries were 92.73% and 85.90%,respectively. The overall recoveries were 91.27% and 81.60%,respectively.

Urine centrifugation recoveries determined using 0-8, 24-48, 72-96, and120-144 hour post-dose samples from the subject ranged between 102% and104%. Urine reconstitution recovery after lyophilization was 94.7% usingpooled samples from a subject.

For pooled fecal samples from 0-48 hours post dose, the extraction,reconstitution, and overall recoveries were 98.48%, 88.80%, and 87.37%,respectively. For pooled fecal samples of 120-168 hours post dose, theextraction, reconstitution, and overall recoveries were 85.85%, 87.69%,and 75.24%, respectively.

The radioactivity recovery from HPLC column for urine sample was 97.05%.

No correction factor was applied to the plasma, urine, and fecalradio-quantitation to account for the recovery.

Metabolite Profiling

In a subject, cabozantinib, compound 9 (cabozantinib sulfate), andcompound 19 (cabozantinib N-oxide) were the major peaks on theradio-chromatograms. Compound 2 (demethylated and sulfated fluoroanilinecleavage product) was the major metabolite in plasma samples after 72hours post-dose. Metabolite compound 7 (fluoroaniline cleavage product)accounted for one of the minor peaks. Metabolite compounds 7, 3(demethyl cabozantinib glucuronide B), 9, and 10 (methyl ester offluoroaniline cleavage product) co-eluted using HPLC Method 1.

Representative human urine metabolite profiles, the radio-chromatograms(using HPLC Method 1) of human urine samples from 0-72 hours, 144-192hours, and 288-336 hours post-dose were collected from a subjectMetabolite compound 6 was the major metabolite in 0-72 hours, 144-192hours, and 288-336 hours post dose pooled urine samples. In addition tocompound 6, metabolite compounds 1, 4, 5, 7, and 19 were observed in thepooled sample of 0-72 hours post dose. Metabolite compounds 1, 4, 5, and7 were observed in the pooled sample of 144-192 hours post dose.Metabolite compounds 1 and 5 were detected in the pooled sample of288-336 hours post dose. Parent compound cabozantinib was not observedin urine samples.

Representative human fecal metabolite profiles, the radio-chromatograms(using HPLC Method 1) of human fecal samples from 0-72 hours, 144-192hours, and 288-336 hours post-dose from a. Parent cabozantinib andmetabolites compound 4, 7, 11, and 15 (including compound 16) wereobserved in the pooled sample of 0-72 hours post dose. Metabolitecompounds 4, 7, 11, 15, 16, and 18 were observed in the pooled sample of144-192 hours post dose. Metabolite compounds 4 and 11 were observed inthe pooled sample of 288-336 hours post dose.

Metabolite Identification Using HPLC/MS Analysis

HPLC/MS analysis of authentic standards using HPLC Method 1 showed thatthe retention times of cabozantinib, fluoroaniline cleavage product(compound 7), sulfate (compound 9), and N-oxide (compound 19) were 20.3,14.4, 16.5, and 23.1 minutes, respectively.

Plasma, urine, and fecal samples were next analyzed by HOPLC/MS, and thecompounds were identified based on their protonated molecular ions andfragmentation patterns.

Metabolite Identification of Cabozantinib and its Metabolites in HumanPlasma

The mass spectrum of the peak at approximately 19.1 minutes in the XICshowed the protonated molecular ions at m/z 502. Its product ion spectrashowed major fragments at m/z 391, 323, and 297, which is consistentwith those of cabozantinib standard. The MS data is summarized in Table1 and 2.

TABLE 1 HPLC Radiochromatogram Retention Times of Metabolites in Samplesfrom Single Oral Dose of [¹⁴C] Cabozantinib Compound HPLC MethodRetention Time (min) Standards 7 1 14.13 9 1 16.45 I 1 20.26 19 1 23.06Plasma 1 1 4.13 2a/2b 1 9.33 4 1 11.87 5 1 12.80 6 1 13.47 7 1 14.13 9 114.67 I 1 18.67 19 1 23.47 Urine 1 1 4.13 4 1 11.87 5 1 12.80 6 1 13.477 1 14.13 19 1 23.47 Feces 4 1 12.67 7 1 13.47 11 1 16.07 15 1 17.87 I 119.60 18 1 21.03 Hamilton Pooled Sample Plasma 9 3 17.36 7 3 19.32 8 319.32 (shoulder) 19 3 25.20 I 3 37.52

TABLE 2 MS Data for Metabolites Using HPLC HPLC HPLC Retention CompoundMethod Time MS (m/z) I 1 19.10 502 19 1 21.85 518 9 1 15.29 518 (loss ofSO₃ from m/z molecular ion m/z at 598) 7 1 13.36 409 2a 1 10.70 (2a)473, 395 (loss of SO₃ from m/z molecular ion m/z at 473) 3 2 15.87 488 82 19.43 488 10 2 33.56 423 5 1 13.00 489 6 1 13.39 393 15 1 17.60 488 161 17.60 518 13 1 16.45 518 12 1 16.45 518 17 1 18.43 518

Kinase Activity of Cabozantinib Metabolites

Kinase Dilution

Kinase Activity was measured and profiled by EMD Millipore according tothe Kinase Profiler Service Assay Protocols Protocol Guide Volume 57.The results are summarized below in Table 3. Inhibition is indicated asIC₅₀ with the following key: A=IC₅₀ less than 50 nM, B=IC₅₀ greater than50 nM, but less than 500 nM, C=IC₅₀ greater than 500 nM, but less than5000 nM, and D=IC₅₀ greater than 5,000 nM. Depending upon thefunctionality about the quinazoline or quinoline, exemplary compounds ofthe invention exhibit selectivity for any of c-Met, KDR, c-Kit, flt-3,and flt-4. Abbreviations for enzymes listed in Table 3 are defined asfollows: c-Met refers to hepatocyte growth factor receptor kinase; RETrefers to RET proto-oncogene kinase; KDR refers to kinase insert domainreceptor tyrosine kinase; flt-1 alpha, fit-3, and flt-4, fins-liketyrosine kinases, representative of the FIX family of receptor tyrosinekinases; and Aurora B MP refers to Aurora B kinase. When a percentage islisted instead of an IC₅₀ value, it indicates percent inhibition at 1uM. Empty cells in the tables indicate lack of data only.

TABLE 3 Kinase Activity Aurora B c-Met RET KDR Flt-1 Flt-3 Flt-4 MP 8ptStd Std Std Alpha Std Std Std Compound (IC50) (IC50) (IC50) (IC50)(IC50) (IC50) (IC50) ID MOLSTRUCTURE (nM) (nM) (nM) (nM) (nM) (nM) (nM)Cabozantinib A A A A A A 16 A A C A B 13 A A C A B 2a ≥50% ≤25% ≤25%≤25% ≤25% ≥25% ≤25% @ 1 μM @ 1 μM @ 1 μM @ 1 μM @ 1 μM @ 1 μM @ 1 μM 2b 9 ≥75% ≥75% ≤25% ≥50% ≥50% ≥75% ≥75% @ 1 μM @ 1 μM @ 1 μM @ 1 μM @ 1 μM@ 1 μM @ 1 μM 19 B B C C  7 D D C C

Metabolite Synthesis and Structural Data 6-Desmethyl Acid

In a vessel, 4-(4-aminophenoxy)-7-methoxyquinolin-6-ol (15.0 g; 53.3mmol), which was prepared according to Scheme 4, and potassium carbonate(29.5 g; 213.3 mmol; 4 equiv) were suspended in THF (210 mL; 14 vol) andwater (90 mL; 6 vol) at 20° C. In a separate vessel, sodium1-(methoxycarbonyl)cyclopropanecarboxylate (17.71 g; 106.6 mmol; 2equiv.) was suspended in THF (90 mL; 6 vol). DMF (120 μL; 3 mol %) wasadded and cooled to less than 15° C. Oxalyl chloride (9.34 mL; 106.6mmol; 2 equiv.) was added over 90 minutes, and the reaction was aged 2hours at 10-15° C. The acid chloride slurry was added to thecabozantinib suspension over 2 hours at 20-25° C. and aged at least 3hours, whereupon HPLC analysis showed greater than 99% conversion to amixture of the mono- and biscarbonylated material. The reaction mixturewas filtered over Celite®, washed with THF (30 mL; 2 vol), and thelayers were separated. 1 M NaOH (150 mL; 10 vol) was added to the upperTHF layer, and the mixture was heated at 40° C. for 1 hour whereuponHPLC analysis showed greater than 99% saponified product. The mixturewas cooled to 25° C., and the upper THF layer was removed. The aqueouslayer was acidified to pH 3-4 with 1 M HCl to precipitate the productand was aged for 1 hour. The precipitate was filtered, washed with water(90 mL, 6 vol), and dried under vacuum (greater than 20 psig) withnitrogen bleed at 50° C. to give a grey to brown powder. ¹H-NMR(DMSO-d₆, 400 MHz) δ 10.8-11.0 (br s, 1H), 10.7 (s, 1H), 8.65 (d, J=6.9Hz, 1H), 7.81 (d, J=9.3 Hz, 2H), 7.67 (s, 1H), 7.58 (s, 1H), 7.32 (d,J=9.3 Hz, 2H), 6.69 (d, J=6.9 Hz, 1H), 4.01 (s, 3H), 2.48-2.53 (m, 4H).MS (ESI−) m/z 393 [M−H]⁻.

6-Hydrogen Sulfate 6-Desmethyl Acid

6-Desmethyl acid (120 mg; 0.30 mmol), potassium hydroxide (118 mg; 2.1mmol; 7 equiv.), and sulfur trioxide trimethyl amine complex (292 mg;2.1 mmol; 7 equiv.) was dissolved in water (3 mL; 25 vol) and heated to70° C. for 2 hours whereupon analysis by HPLC showed greater than 99%conversion. The reaction mixture was then cooled in an ice bath andacidified dropwise with 1 N aq. H₂SO₄ to approximately pH I. The slurrywas aged at 25° C. for 1 hour, filtered, washed with water (3 mL; 25vol), and deliquored. The wet cake was then washed with acetone (3 mL;25 vol) and dried at 35° C. under vacuum (greater than 20 psig) withnitrogen bleed for 24 hours to give a beige powder.

Alternatively, 6-desmethyl acid (120 mg; 0.30 mmol) was suspended inMeCN (50 vol, 6 mL), and triethylamine (1.27 mL, 9.12 mmol, 30 equiv.)was added and then cooled in an ice bath. Chlorosulfonic acid (101 μL,1.52 mmol, 5 equiv.) was added dropwise, and the reaction was thenheated to 70° C. for 1 hour whereupon analysis by HPLC showed greaterthan 98 percent conversion. The reaction mixture was then cooled in anice bath for 2 hours in which a precipitate was formed. The precipitatewas removed with filtration, rinsing with cold MeCN (50 vol). The MeCNsolution was then concentrated to approximately 20 vol (approximately 2mL) and quenched with 100 vol 1N HCl and cooled in an ice bath to give afine precipitate that was filtered, washed with 50 vol water and 50 volacetone, and dried at 35° C. under vacuum (greater than 20 psig) withnitrogen bleed for 24 hours to give a beige powder. ¹H-NMR (DMSO-d₆, 400MHz) δ 10.8 (s, 1H), 8.83 (d, J=5.9 Hz, 1H), 8.5 (s, 1H), 7.85 (d, J=8.5Hz, 2H), 7.52 (s, 1H), 7.40 (d, J=8.5 Hz, 2H), 6.84 (d, J=5.9 Hz, 1H),4.04 (s, 3H), 3.20-3.70 (br s, 1H), 1.39-1.48 (br s, 4H). MS (ESI−) m/z473 [M−H]⁻, 236.

Ortho-Hydroxy-Cabozantinib

A flask was charged with the carboxylic acid (0.84 g; 2.1 mmol), THF(1.2 mL), and DMF (5 μL), and cooled to 15° C. To this slurry was addedoxalyl chloride (0.17 mL; 2.1 mmol) dropwise over approximately 20minutes. After 2 hours, the acid chloride slurry was added to anothervessel containing a stirred suspension of the aniline (0.2 g, 1.6 mmol),potassium carbonate (0.63 g, 4.6 mmol) in THF (2.8 mL), and water (1 mL)over approximately 15 minutes. After 3 hours, HPLC analysis showedcomplete conversion to the product. Stirring was stopped, the loweraqueous layer was removed, and water (30 mL) was added to precipitatethe product. The product was then collected by filtration and washedwith 1:1 THF-water solution (2×10 mL) to afford a pale grey solid. Itwas then further purified by flash chromatography on silica gel usingmethanol/dichloromethane as the mobile phase.

Alternatively, a suspension of the carboxylic acid (4.08 g; 10 mmol),aniline (1.52 g; 12 mmol), and triethylamine (2.7 mL; 20 mmol) inacetonitrile (100 mL) was treated with EDAC (2.30 g; 12 mmol) and HOBt(0.5 g; 3 mmol). The slurry was stirred overnight at room temperature,and the reaction progress was monitored by HPLC. At the end of thereaction, 150 mL of water was added, and the precipitated product wascollected by filtration, washed with water, and then purified by flashchromatography. ¹H-NMR (DMSO-d₆, 400 MHz) δ 10.46 (br s, 1H), 10.29 (brs, 1H), 10.0 (br s, 1H), 8.47 (d, 1H), 7.92 (dd, 1H), 7.73 (dd, 2H),7.51 (s, 1H), 7.40 (s, 1H), 7.28 (dd, 2H), 6.68 (dd, 1H), 6.62 (dt, 1H),6.45 (d, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 1.60-1.55 (m, 4H). ¹³C NMR(DMSO-d₆, 100 MHz) δ 169.82, 167.67, 159.91, 157.51, 152.58, 149.97,149.35, 149.09, 148.98, 148.86, 146.49, 135.72, 123.00, 122.97, 122.91,122.43, 121.30, 115.17, 107.86, 105.10, 104.87, 103.16, 102.43, 102.19,99.08, 55.74, 55.71, 55.66, 30.02, 16.51.

MS (APCI+) m/z 518.3 [M+H]⁺, 500.3.

Cabozantinib-Hydroxysulfate

A suspension of the hydroxy-cabozantinib (0.95 g; 1.9 mmol) in THF (20mL) was added triethylamine (5 mL; 36 mmol), and cooled to below 5° C.Chlorosulfonic acid (1 mL; 15 mmol) was added dropwise such that thetemperature remained below 10° C., over approximately 15 minutes. Afterstirring overnight at room temperature, HPLC analysis showedapproximately 5 percent of starting material remaining. The reactionmixture was treated with aqueous 1 N HCl (25 mL). The precipitatedproduct was collected by filtration, washed with water (4×25 mL), anddried under vacuum to yield an off-white solid (937 mg; 82 percent crudeyield). Analysis by AN-HPLC showed that the product was 90.8% pure, themajor impurity being the starting material. The product was purified togreater than 99 percent (AN-HPLC) by preparative HPLC on a C18 column,using aqueous ammonium acetate/acetonitrile mobile phase system. ¹H-NMR(DMSO-d₆, 400 MHz) δ 10.39 (s, 1H), 9.69 (s, 1H), 8.81 (d, 1H), 7.95(dd, 1H), 7.85 (d, 2H), 7.77 (s, 1H), 7.51 (s, 1H), 7.11 (s, 1H), 7.08(dd, 1H), 6.93 (dd, 1H), 6.45 (d, 1H), 4.05 (s, 3H), 4.04 (s, 3H), 1.53(s, 4H). MS (ESI−) m/z 596.0 [M−H]⁺.

Meta-Hydroxy-Cabozantinib

A flask was charged with the carboxylic acid (0.84 g; 2.1 mmol), THF(1.2 mL), and DMF (5 μL.), and cooled to 15° C. To this slurry was addedoxalyl chloride (0.17 mL; 2.1 mmol) dropwise over approximately 20minutes. After 2 hours, the acid chloride slurry was added to anothervessel containing a stirred suspension of the aniline (0.2 g, 1.6 mmol),potassium carbonate (0.63 g, 4.6 mmol) in THF (2.8 mL), and water (1 mL)over approximately 15 minutes. After 90 minutes, HPLC analysis showedcomplete conversion to the product. Stirring was stopped, and the loweraqueous layer was removed and extracted with ethyl acetate (15 mL). Theorganic layers were combined, dried over anhydrous MgSO₄, filtered, andconcentrated to yield a brown solid. The solid was then further purifiedby flash chromatography on silica gel using ethyl acetate/heptane as themobile phase. ¹H-NMR (DMSO-d₆, 400 MHz) δ 10.15 (br s, 1H), 9.96 (br s,1H), 9.89 (br s, 1H), 8.46 (d, 1H), 7.76 (d, 1H), 7.50 (s, 1H), 7.41 (d,2H), 7.39 (s, 1H), 7.22 (d, 2H), 7.07-6.98 (m, 2H), 6.42 (d, 1H), 3.94(s, 3H), 3.93 (s, 3H), 1.46 (br s, 4H). ¹³C NMR (DMSO-d₆, 100 MHz) δ168.27, 167.95, 160.02, 152.56, 149.48, 149.33, 148.86, 148.56, 146.46,146.21, 144.52, 144.39, 136.45, 135.33, 135.31, 122.23, 121.22, 115.63,115.44, 115.15, 111.29, 111.23, 110.26, 107.85, 103.04, 99.08, 55.73,55.71, 31.66, 15.40. MS (APCI+) m/z 518.3 [M+H]⁺, 502.3.

Cabozantinib N-Oxide

A flask was charged with cabozantinib (3.21 g; 6.4 mmol), acetic acid(32.1 mL), and sodium perborate tetrahydrate (1.98 g, 12.8 mmol) andheated to 65° C. and stirred overnight. After 24 hours, HPLC analysisshowed about 38:62 starting material:product. More oxidant (1.98 g; 12.8mmol) was added, and heating continued overnight. Solvents were removedunder vacuum, and the residue was purified by flash chromatography usingdichloromethane-methanol gradient (dichloromethane to 10%methanol-dichloromethane) to obtain 0.95 g of the product as a whitesolid. ¹H-NMR (DMSO-d₆, 400 MHz) δ 10.20 (br s, 1H), 10.08 (br s, 1H),8.28 (d, 1H), 7.90 (s, 1H), 7.74 (d, 2H), 7.64 (dd, 2H), 7.48 (s, 1H),7.23 (d, 2H), 7.15 (t, 2H), 6.45 (d, 1H), 3.97 (s, 311), 3.94 (s, 311),1.47 (br s, 4H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.11, 168.18, 168.13,159.49, 157.09, 153.34, 150.72, 150.57, 149.98, 137.41, 136.32, 135.24,135.21, 134.06, 122.44, 122.36, 122.19, 120.65, 117.23, 11.17, 114.95,104.37, 100.34, 99.12, 56.09, 56.03, 31.59, 15.42. MS (APCI+) m/z 518.3[M+H]⁺.

1-[4-(6,7-Dimethoxy-quinolin-4-yloxy)-phenylcarbamoyl]-cyclopropanecarboxylic acid

To the cyclopropyl di-carboxylic acid (449 mg, 3.45 mmol) in THF (3.5mL) was added TEA (485 μL, 3.45 mmol). The resulting solution wasstirred at room temperature under a nitrogen atmosphere for 40 minutesbefore adding thionyl chloride (250 μL, 3.44 mmol). The reaction wasmonitored by LCMS for the formation of mono acid chloride (quenched thesample with MeOH and looked for corresponding mono methyl ester). After3 hours stirring at room temperature,4-(6,7-dimethoxy-quinolin-4-yloxy)-phenylamine (1.02 g, 3.44 mmol) wasadded as a solid, followed by more THF (1.5 mL). The reaction continuedto stir at room temperature for 16 hours. The resulting thick slurry wasdiluted with EtOAc (10 mL) and extracted with IN NaOH. The biphasicslurry was filtered, and the aqueous phase was acidified withconcentrated HCl to pH or approximately 6 and filtered. Both solids werecombined and washed with EtOAc, then dried under vacuum. The desiredproduct1-[4-(6,7-dimethoxy-quinolin-4-yloxy)-phenylcarbamoyl]-cyclopropanecarboxylicacid, was obtained (962 mg, 68.7 percent yield, 97 percent pure) as awhite solid. ¹H NMR (D₂O/NaOH): 7.97 (d, 1H), 7.18 (d, 2H), 6.76 (m,4H), 6.08 (d, 1H), 3.73 (s, 3H), 3.56 (s, 3H), 1.15 (d, 4H).

The foregoing disclosure has been described in some detail by way ofillustration and example for purposes of clarity and understanding. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications can be made while remainingwithin the spirit and scope of the invention. It will be obvious to oneof skill in the art that changes and modifications can be practicedwithin the scope of the appended claims Therefore, it is to beunderstood that the above description is intended to be illustrative andnot restrictive. The scope of the invention should, therefore, bedetermined not with reference to the above description, but shouldinstead be determined with reference to the following appended claims,along with the full scope of equivalents to which such claims areentitled.

1. An isolated metabolite of cabozantinib or a pharmaceutically acceptable salt thereof.
 2. The isolated metabolite of claim 1 which is a compound selected from:

wherein GA is a glucuronic acid moiety, or a pharmaceutically acceptable salt thereof.
 3. The isolated metabolite of claim 2, which is selected from:

or a pharmaceutically acceptable salt thereof.
 4. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 5. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 6. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 7. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 8. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 9. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 10. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 11. The isolated metabolite of claim 2 which is

or a pharmaceutically acceptable salt thereof.
 12. A method of treating a disease treating diseases or disorders associated with uncontrolled, abnormal, and/or unwanted cellular activities, the method comprising administering, to a mammal in need thereof, a therapeutically effective amount of a compound of claims 1-11, or a pharmaceutically acceptable salt thereof.
 13. A pharmaceutical composition comprising:

or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.
 14. The composition of claim of claim 13, which is suitable for oral administration.
 15. A compound which is selected from:

wherein GA is a glucuronic acid moiety, or a pharmaceutically acceptable salt thereof.
 16. The compound of claim 15, which is selected from:

or a pharmaceutically acceptable salt thereof.
 17. A method for identifying a metabolite of cabozantinib, comprising: administering cabozantinib to a mammal; detecting or measuring a level or concentration of a metabolite of N-(4-{[6,7-bis(methyloxy)quinolin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide in a mammal in tissues or bodily fluids of the mammal; wherein the metabolite is selected from the group consisting of:

wherein GA is a glucuronic acid moiety.
 18. The method of claim 17, wherein the metabolite is selected from:


19. The method according to claim 17, wherein the bodily fluids are selected from the group consisting of plasma, bile, urine, and feces. 